Project Summary Antibodies are an essential component of adaptive immunity, providing protection from microbial infection. However, antibodies can also contribute to disease in the context of autoantibodies and alloantibodies. Although often considered an endpoint of humoral immunity, it has long been appreciated that antibodies can also modulate the development of adaptive immune responses in an antigen specific fashion and as a function of the antigens they recognize. This general phenomenon has been named antibody mediated immune suppression (AMIS), and has been observed in a wide variety of settings, including immunity to microbial pathogens, vaccination, transplantation and transfusion. Perhaps the most well known case of AMIS in humans is the administration of anti-RhD as a therapeutic to prophylax against alloimmunization during pregnancy and/or delivery, which has been a widely successful approach to decrease hemolytic disease of the fetus and newborn. Indeed, anti-RhD remains one of the only antigen specific immune therapies, allowing prevention of immunization to a specific target without general immunosuppression. Thus, the practical potential of harnessing AMIS for the treatment of human disease is well established. However, despite the success of anti-RhD, the mechanism of immune suppression remains poorly understood. Moreover, several studies have reported that anti-RhD paradoxically enhances alloimmunization in some settings, underscoring our poor understanding of anti-RhD mechanisms of action. Lack of mechanistic understanding has also hampered attempts to make monoclonal anti-RhD, with some monoclonals suppressing immunity whereas others enhance. The current application makes use of an innovative preclinical mouse model of alloimmunization to RBCs to test a series of distinct mechanistic hypotheses regarding AMIS effects, under the central hypothesis that the IgG subtype of an antibody is a key factor in its ability to regulate immunity. Indeed, preliminary data demonstrates that IgG subtype determines if an antibody is immune suppressing or immune enhancing. The proposal uses a combination of novel tools and murine strains, as well as steps to humanize the murine model. In this context, 3 specific aims are proposed. Specific aim 1: Mechanisms by which IgG2a anti-RBC antibodies enhance alloimmunization to RBCs. Specific aim 2: Mechanisms by which IgG1 anti- RBC antibodies suppresses humoral alloimmunization to RBCs. Specific Aim 3: Testing effects and mechanisms of anti-RBC antibodies on alloimmunization in a humanized mouse model. The long-term goals of this project are to generate novel mechanistic understanding that will provide the conceptual basis for future human trials to refine monoclonal anti-RhD, to provide a basis for how AMIS like effects can be applied to other new therapeutics in different areas, and to generate a basic understanding of how antibodies regulate humoral immunity in general. Such knowledge is relevant to understanding processes of humoral immunity as it relates to adaptive anti-microbial immunity, pathogenesis of humoral autoimmunity, and immune-mediated diseases.